1. Field of the Invention
The present invention provides expression vectors comprising internal promoters that can be used for expressing proteins of interest. In one embodiment, the present invention provides retroviral vectors comprising an enhancer-deleted U3 region.
2. Background Art
Gene transfer involves the transfer of genetic material to a cell, usually for transcription and expression. The method is ideal for protein expression as well as for therapeutic purposes. Various transfer methods are known, such as DNA transfection and viral transduction. Virally-mediated gene transfer is attractive due to the efficiency of transfer and high levels of transgene expression, as well as the potential for targeting particular receptors and/or cell types if needed through natural affinity or pseudotyping.
In particular, retroviral vectors are useful for longer term expression due to their ability to integrate into the cellular genome. Murine leukemia virus-based (MLV) vectors are the most common retroviral vector, with many backbone plasmids and packaging cell lines available to suit most applications (See e.g., Miller and Buttimore, Mol. Cell. Biol. 6:2895 (1986)). Like all “simple” retroviruses, e.g. retroviruses that only encode structural and enzymatic viral proteins and do not utilize viral accessory proteins, MLV vectors can only integrate into dividing cells. Other simple retroviruses potentially suitable for use as vectors include other members of the mammalian C-type viruses (e.g., murine stem cell virus, Harvey murine sarcoma virus and spleen necrosis virus), B type viruses (e.g., mouse mammary tumor virus), and D type viruses (e.g., Mason Pfizer monkey virus). Other retroviruses suitable for use as a retroviral vector of the invention include avian retroviruses (e.g., Rous sarcoma virus), spumaviruses (e.g., foamy viruses), and the HTLV-BLV viruses (e.g., HTLV-1).
Lentiviruses are a subgroup of retroviruses that express viral accessory proteins and are capable of infecting and integrating into non-dividing, as well as dividing, cells. Vectors derived from lentiviruses are ideal tools for delivering exogenous genes to target cells because of their ability to stably integrate into the genome of dividing and non-dividing cells and to mediate long-term gene expression (Gilbert et al., Somat. Cell Mol. Genet. 26:83 (2001); Mitrophanous et al., Gene Ther. 6:1808 (1999); Naldini et al., Science 272:263 (1996); Sauter et al., Somat. Cell Mol. Genet. 26:99 (2001)).
Lentiviruses have been isolated from many vertebrate species including primates, e.g., human and simian immunodeficiency viruses (HIV-1, HIV-2, SIV), as well as non-primates, e.g., feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), equine infectious virus (EIAV), caprine arthritis encephalitis virus (CAEV) and the visna virus. Of these, HIV and SIV are presently best understood. Among non-primate lentiviral vectors, vectors derived from FIV (Curran et al., Curr. Top. Microbiol. Immunol. 261:75 (2002)) and EIAV (U.S. Patent Application No. 2001/0044149) are best characterized.
There are two major safety aspects that have received considerable attention in the context of retroviral gene therapy, regardless of whether the vector is based on murine leukemia virus (MLV) or lentivirus. Specifically, they are the presence of replication competent retrovirus (RCR) and the incidence of insertional mutagenesis. The former problem has greatly been improved by the development of a minimum sized retroviral vector that contains no overlapping viral sequences between the vectors and the packaging genome. However, the latter possibility has recently raised serious concerns, mainly because of the three leukemia cases found in the X-SCID human trial (Hacein-Bey-Abina et al., Science 302:415 (2003)). The retrospective analysis of the first two leukemia cases revealed that the leukemia probably resulted from the retroviral integration into the chromosome and the subsequent activation of the LMO2 gene, located in close proximity to the integration site, by the long terminal repeat (LTR). Although it was argued that this vector-mediated tumorigenesis might be restricted to the X-SCID gene therapy case due to the particular nature of this disease and its gene, it is now clear that the safety of retroviral vectors needs further improvement to become a viable form of therapeutics in the real world.
There have been several approaches for reducing the probability of vector-mediated tumorigenesis. One approach is to remove the U3 region of the LTR (Yu et al., Proc. Natl. Acad. Sci. USA 83:3194 (1986); Hawley et al., Proc. Natl. Acad. Sci. USA 84:2406 (1987); Yee et al., Proc. Natl. Acad. Sci. USA 84:5197 (1987)). The retroviral LTR consists of U3, R, and U5 regions, and the U3 region contains the enhancer and promoter sequence that control gene expression (Sun et al., J. Virol. 69:4941 (1995); Wahlers et al., Mol. Ther. 6:313 (2002)). Therefore, the insertional activation by a vector can be reduced by removing the U3 region. In that case, an additional promoter should be supplied to the vector to drive the expression of the target gene because the U3-deleted vector no longer contains the promoter sequence in the LTR.
As discussed previously, the U3-inactivated retroviral vector needs an internal promoter for the expression of target gene. One of the most frequently used internal promoters in retroviral vectors is the human cytomegalovirus (HCMV) immediate-early (IE) promoter (Jaalouk et al., Virol. J. 3:27 (2006); pQCXIN available from BD Biosciences) or related ones such as CA (HCMV IE enhancer/chicken β-actin promoter) (Ramezani et al., Mol. Ther. 14:245 (2006)). However, the HCMV IE promoter is known to be rapidly inactivated in primary human cells, while it does not work for certain genes (Herweijer et al., J. Gene Med. 3:280 (2001)). Thus, commonly used promoters have been shown to decrease expression of heterologous genes, be inactive in certain cell types, and potentially activate LTR-driven transcription, all of which decrease the safety and efficacy of the retroviral vector.
Finally, U3-inactivated retroviral vectors have been associated with very low titers due to promoter suppression by commonly-used promoters, such as CMV and SV40, which reduce transcription of genomic RNA for packaging (Jaalouk et al., Virol. J. 3:27 (2006)) Indeed, MLV based U3-deleted vectors have been associated with titers up to four orders of magnitude less than the comparable MLV vector with intact U3 regions (Olson et al., J. Virol. 68:7060 (1994)). Thus, it is surprising to find promoters that are capable of both driving high levels of heterologous gene transcription as well as enabling high viral titers to be produced. Therefore, new promoters are needed to be developed for use as an internal promoter in the retroviral vector.